Studies on the site(s) of oxygen-linked binding of inorganic anions to hemoglobin will be continued. Through the use of a hemoglobin derivative specifically carbamylated on the NH2-termini of the Beta-chains (alpha 2 Beta C over 2), we had gained evidence that excluded Val-l of the Beta-chains as a primary, oxygen-linked binding site for inorganic anions. We postulated that lysine-82 of the Beta-chains was the important residue for this binding. We plan to test that proposal by similar studies on hemoglobin Providence, which contains an asparagine or an aspartic acid residue at position 82. The observation that Val-l (alpha) is carbamylated about 2.5 times more rapidly in the deoxy compared with the oxy state is under further study. We plan to develop a rapid and simple method to measure the rate constant for the carbamylation of this residue, and to apply this technique to determination of the amount of R or T structure for a given hemoglobin in the presence of various heterotropic regulators. Since the clinical application of sodium cyanate to sickle-cell disease now seems doubtful because of accompanying side effects, we plan to use the knowledge that we have gained on the mechanism of action of cyanate on hemoglobin S to design other modifiers of the NH2-terminal residues of the alpha- and of the Beta-chains that might increase the oxygen affinity or decrease the gelling of deoxyhemoglobin S. BIBLIOGRAPHIC REFERENCES: Nigen, A.M., and Manning, J.M. The Interaction of Anions with Hemoglobin Carbamylated on Specific NH2-Terminal Residues. J. Biol. Chem. 250, 8248-8250 (1975). Nigen, A.M., Manning, J.M., Peterson, C.M., and White, J.M. Bioavailability of Sodium Cyanate in Patients with Sickle Cell Disease and the Lack of Inhibition In Vitro of Globin Synthesis at In Vivo Concentrations of Cyanate. J. Pharmacol. Exp. Therap. 195, 333-339 (1975).